Given that proteins regulate almost every biological process, the exploration of protein interaction networks is expected to have major impacts on the understanding of biological systems, disease mechanisms and drug discovery. As a result, development of technologies to identify binding proteins is of major importance in functional proteomics. Yeast two-hybrid system (Y2H) and mass spectrometry coupled with tandem affinity purification (MS-TAP) are two examples. However, these systems are limited by technical complexity, instrument requirements, labor and time commitments. Furthermore, their application is narrowly restricted to bait proteins, but not to other biological molecules.
Since its first description in 1985, phage display has been widely used to identify bait-binding antibodies (Abs) or short peptides from antibody or random peptide libraries. However, phage display with cDNA libraries is rare and inefficient. Although antibody libraries with predictable reading frames can be conveniently fused to the N-terminus of filamentous phage gene III capsid protein (pIII) without reading frame shift, a cDNA library with unpredictable reading frames and stop codons may interfere with pIII expression. To circumvent the problem, various strategies of C-terminal display have been explored. However, C-terminal display cannot ensure that the cDNA library is expressed in the correct reading frames. Unlike Y2H, non-open-reading-frame (non-ORF) phage clones encoding unnatural short peptides tend to outgrow ORF clones through multiple rounds of selection and amplification. Consequently, most of identified phage clones encode out-of-frame unnatural short peptides, rather than real proteins, with minimal implication in protein biological networks. It, thus, remains a daunting challenge for the technology to be applied to functional proteomics with an efficiency comparable to Y2H and MS-TAP.